A WRKY transcription factor from Withania somnifera...

AWRKY transcription factor fromWithania somnifera regulatestriterpenoid withanolide accumulation and biotic stress tolerancethrough modulation of phytosterol and defense pathways

Anup Kumar Singh1*, Sarma Rajeev Kumar1*, Varun Dwivedi1*, Avanish Rai1, Shaifali Pal2, Ajit K. Shasany2 and

Dinesh A. Nagegowda1

1Molecular Plant Biology and Biotechnology Lab, CSIR-Central Institute of Medicinal and Aromatic Plants Research Centre, Allalasandra, GKVK Post, Bengaluru 560065, India;

2Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India

Author for correspondence:Dinesh A. NagegowdaTel: +91 80 28460563

Email: [email protected]

Received: 30 January 2017Accepted: 6 May 2017

New Phytologist (2017)doi: 10.1111/nph.14663

Key words: overexpression, phytosterols,regulation, triterpenoids, virus-induced genesilencing (VIGS),Withania somnifera, with-anolides, WRKY transcription factor.

Summary

� Withania somnifera produces pharmacologically important triterpenoid withanolides that

are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be

elucidated.� A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera

(WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally charac-

terized employing virus-induced gene silencing and overexpression studies combined with

transcript and metabolite analyses, and chromatin immunoprecipitation assay.� WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol

pathway genes with corresponding reduction in phytosterols and withanolides in

W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera

(phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover,

WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding

squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid

pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the toler-

ance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco

led to improved biotic stress tolerance.� Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phy-

tosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its

importance as a metabolic engineering tool for simultaneous improvement of triterpenoid

biosynthesis and plant defense.

Introduction

Triterpenoids are ubiquitous in eukaryotes as primary sterolmetabolites, which are important components of membranes andsignaling (as steroidal hormones) (Moses et al., 2013; Kemenet al., 2014). Plants also synthesize specialized triterpenoids thatare often accumulated as conjugates with macromolecules andserve as defense molecules against biotic and abiotic stresses(Augustin et al., 2011; de Costa et al., 2013). Triterpenoids alsohave a wide range of commercial applications because of theirbeneficial properties for humans. Withania somnifera (Ashwa-gandha, winter cherry or Indian ginseng), a solanaceous plant,accumulates pharmacologically important steroidal lactonetriterpenoids called withanolides, which are termed22-hydroxyergostan-26-oic acid-26,22-lactones (Dhar et al.,2014). W. somnifera extracts are reported to possess various

pharmacological activities, including antitumour, anti-inflammation, cardioprotective and neuroprotective properties(Mirjalili et al., 2009; Sehgal et al., 2012). So far > 40 withano-lides have been isolated from this plant in which withaferin Aand withanolide D have been reported to inhibit angiogenesis,Notch-1 and NFjB in cancer cells, and induce apoptosis inbreast cancer cells (Kaileh et al., 2007; Koduru et al., 2010;Hahm et al., 2011). Although extensive work has been carriedout in W. somnifera with respect to metabolic profiling and phar-macological activities, there is a lack of information related topathway steps in withanolide biosynthesis. Withanolides arereported to be formed via either the cytosolic mevalonate (MVA)pathway or the plastidial methylerythritol phosphate (MEP)pathway, which provide isopentenyl diphosphate (IPP) anddimethylallyl diphosphate (DMAPP) for the synthesis of triter-penoid pathway intermediates (Sangwan et al., 2008; Jadaunet al., 2017). ‘Head-to-tail’ condensation of IPP and DMAPPforms C15 farnesyl diphosphate (FPP), which is further*These authors contributed equally to this work.

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condensed by squalene synthase (SQS) to form the linear C30

triterpenoid precursor, squalene. Subsequently, squalene epoxi-dase (SQE) condenses squalene to form 2,3-oxidosqualene,which undergoes cyclization by specific oxidosqualene cyclases(OSCs) leading to tetra- or pentacyclic structures (Phillips et al.,2006; Nes, 2011). Later, cycloartenol synthase (CAS) converts2,3-oxidosqualene to cycloartenol, which acts as the committedprecursor for plant sterols. It is believed that intermediates syn-thesized via the universal sterol pathway act as precursors for thebiosynthesis of withanolides. These precursors are proposed toundergo various biochemical transformations like hydroxylationand glycosylation reactions, leading to the formation of variouswithanolides (Lockley et al., 1976; Sangwan et al., 2008; Singhet al., 2014). As current knowledge of withanolides biosyntheticgenes is lacking, use of transcription factors (TFs) that are oftencapable of coordinating transcription of multiple biosyntheticpathway genes holds great promise for improving the productionof withanolides, either at the whole-plant level or in culture sys-tems. By manipulating the expression of a single TF, it is possibleto affect the expression of several coordinately regulated biosyn-thetic genes, leading to increased metabolite production (Grote-wold, 2008).

WRKY TFs are generally known for their role in the regulationof stress tolerance in plants (Schluttenhofer & Yuan, 2015).However, accumulating evidence suggests that certain WRKYsregulate the production of secondary metabolites by regulatinggenes involved in few or many steps of a metabolic pathway.WRKYs regulating the biosynthesis of all three major classes ofsecondary metabolites (terpenes, phenylpropanoids and alka-loids) have been identified (Schluttenhofer & Yuan, 2015). Forinstance, WRKY TFs are reported to regulate the biosynthesis ofspecialized monoterpenes in tomato (Spyropoulou et al., 2014),sesquiterpenes such as artemisinin in Artemisia annua (Ma et al.,2009; Chen et al., 2016), gossypol in Gossypium arboreum (Xuet al., 2004) and capsidiol in tobacco (Ishihama et al., 2011),diterpenes momilactone A in rice (Akagi et al., 2014) and pacli-taxel in Taxus chinensis (Li et al., 2013), and polyterpenes in rub-ber tree (Zhang et al., 2012; Wang et al., 2013). In the case oftriterpenoids, although PqWRKY1 from Panax quinquefolius reg-ulated triterpenoid pathway genes such as FPPS, SQS and SQE inArabidopsis, its effect on triterpenoid metabolites was not studied(Sun et al., 2013). In addition to WRKYs, TFs of basic helix–loop–helix (bHLH) and ethylene-responsive factor (ERF) classhave been recently shown to regulate triterpenoid biosynthesis inMedicago truncatula and tomato, respectively (Thagun et al.,2016; C�ardenas et al., 2016; Mertens et al., 2016).

It was reported that nitrogen starvation leads to drastic reduc-tion of withanolides in W. somnifera (Devkar et al., 2015). In ourrecent study, nitrogen treatment of W. somnifera enhanced theaccumulation of phytosterols and withaferin A which correlatedwith higher expression of sterol pathway genes. Further, it wasshown that, among different classes of TFs analyzed (WRKY,MYB, bHLH and bZIP), a particular TF WsWRKY1 (W. som-nifera WRKY1) was highly induced in nitrogen-treated samples(Pal et al., 2017). Here, we present the functional characteriza-tion of WsWRKY1 which shows its important role in regulation

of triterpenoid biosynthesis and defense in W. somnifera. Further,we demonstrate the positive regulatory role of WsWRKY1 in phy-tosterol biosynthesis and defense in a heterologous host system.Our results show that the WsWRKY1 regulates withanolide accu-mulation via transcriptional modulation of the phytosterol path-way and also controls defense response.

Materials and Methods

Plant material, phytohormone treatment and tissuecollection

Withania somnifera (L.) Dunal cv Poshita (National Gene Bank,CSIR-CIMAP, India) seeds were germinated in plastic pots con-taining soil mixture and grown in a growth room at 22–25°Cwith a 16 : 8 h, light : dark photoperiod. For virus-induced genesilencing (VIGS) experiments, 3-wk-old four-leaf staged seedlingswere used for agroinfiltration. For phytohormone treatments,third developmental leaves of W. somnifera were plucked from10-wk-old plants kept in Petri dishes containing solutions ofsalicin (a derivative of salicylic acid, SA; 1.5 mM) or methyl jas-monate (MeJA; 500 lM) with respective controls. To achieve therequired concentration of phytohormones, salicin was dissolvedin deionized water, whereas MeJA was dissolved in dimethyl sul-foxide (DMSO; 22.8 ll corresponding to 4.3M was added to977.2 ll of DMSO) and diluted with deionized water. Sampleswere harvested at different time (h) intervals and stored at �80°Cuntil further use. For tissue-specific expression analysis, roots,stems and leaves were collected separately from 6-wk-old plants,frozen in liquid nitrogen and stored at �80°C until further use.For generation of transgenic plants Nicotiana tabacum L. cv Xan-thi was used.

Phylogenetic analysis

All WRKY sequences reported to be involved in secondarymetabolism and some WRKYs that have been shown to be regu-lating stress tolerance were used for constructing the tree. A phy-logenetic tree was constructed with the neighbor-joining methodusing default settings of MEGA v.6 (Tamura et al., 2013). Boot-strap values were calculated from 1000 replicates.

Subcellular localization

The open reading frame (ORF) of WsWRKY1 was amplified byPCR using leaf cDNA with gene-specific forward and reverseprimers consisting of XbaI/BamHI sites (Supporting InformationTable S1). The resulting PCR product was cloned into pJET1.2/vector for sequence confirmation followed by subcloning into theXbaI/BamHI sites of p326–sGFP vector yielding the WsWRKY1-GFP construct (Fig. S1). Plasmids p326::WsWRKY1-sGFP andp326::sGFP were introduced into Catharanthus roseus protoplastsusing PEG-mediated transformation as described previously (Yooet al., 2007; Nagegowda et al., 2008). The transformed cells werethen observed using a fluorescence microscope (DMI 3000B;Leica, Wetzlar, Germany).

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Construction of VIGS and overexpression vectors

Plasmids pTRV1 and pTRV2 (described in Liu et al., 2002) pro-cured from TAIR (www.arabidopsis.org) were used for generat-ing the VIGS construct. A 540 bp WsWRKY1 fragment wasPCR-amplified from leaf cDNA using gene-specific primers(Table S1). The resulting PCR product was cloned into thepJET1.2/vector for sequence confirmation, followed by sub-cloning into the EcoRI site of the pTRV2 vector yieldingpTRV2::WsWRKY1 construct (Fig. S1). For generation of theoverexpression construct, the ORF of WsWRKY1 was PCR-amplified using leaf cDNA with specific forward and reverseprimers (Table S1). The amplified fragment was cloned into thepJET1.2/vector and sequences were confirmed by nucleotidesequencing. Later, the WsWRKY1 fragment was restriction-digested and subcloned into the XbaI and SacI sites of the pBI121binary vector under the control of the 35S promoter ofCauliflower mosaic virus (CaMV) to form the pBI121::WsWRKY1 construct (Fig. S1). Generated VIGS and overexpres-sion constructs were introduced into Agrobacterium tumefaciensstrain GV3101 by the freeze–thaw method.

VIGS and transient overexpression ofWsWRKY1

Virus-induced gene silencing experiments were carried out by leafinfiltration according to Singh et al. (2015). As VIGS is prone tooff targets, the sequence region unique to WsWRKY1 was chosenfor generating the VIGS construct. The specificity of WsWRKY1silencing was further confirmed by measuring the expression oftwo other similar W. somnifera WRKY genes that showed noeffect on these genes (Fig. S2). Transient overexpression was per-formed in W. somnifera and tomato (Solanum lycopersicum) leavesaccording to Kumar et al. (2015). Briefly, overnight grownAgrobacteria cultures were pelleted and resuspended in infiltrationbuffer (50 mM MES, pH 5.6, 2 mM Na3PO4, 0.5% glucose,and 100 lM acetosyringone) to a final OD600 of 0.15–0.2. Thesuspension was further incubated at 28°C for 4 h before infiltra-tion. Agrobacteria cultures were infiltrated into the first leaf pairand plants were maintained in the dark for 48 h. Leaves were har-vested and stored at �80°C until further use for transcript andmetabolite analyses.

Generation of transgenic tobacco plants

Plasmids pBI121::WsWRKY1 and pBI121 were used to gener-ate transgenic tobacco by the Agrobacterium-mediated leaf disccocultivation method (Horsch et al., 1985). After three roundsof selection on MS (Murashige & Skoog, 1962) medium sup-plemented with kanamycin (100 mg l�1), regenerated shootswere excised and transferred to rooting media. Plantlets withwell-established root systems were hardened for 2 wk in soilritemix (Keltech Energies Ltd, Bengaluru, India) and then trans-ferred to the glasshouse. T1 seeds from individual transfor-mants were selected on MS medium with kanamycin(50 mg l�1), and the resistant seedlings were transferred to soil.Transformed plants were screened by PCR and leaves from

positive lines were used for transcript and metabolite analyses,and bacterial growth assay.

RNA extraction and RT-qPCR analyses

Total RNA extraction, cDNA synthesis and quantitative reversetranscription-polymerase chain reaction (RT-qPCR) were carriedout as described earlier (Rai et al., 2013). 18S rRNA forW. somnifera, and EF-1a for N. tabacum and tomato served asendogenous controls in RT-qPCR. For quantification ofWsWRKY1transcripts, primers (Table S1) were designed outside the generegion cloned in pTRV2. RT-qPCR conditions were as follows:94°C for 10min for one cycle, followed by 40 cycles of 94°C for15 s and 60°C for 1min. Fold-change differences in gene expressionwere analyzed using the comparative cycle threshold method.

Squalene and withanolide analysis

Squalene was extracted according to Qin et al. (2010) using20 mg DW leaf tissue. For squalene analysis, the extracted residuewas dissolved in 20 ll of chloroform/methanol (7 : 3, v/v) andwas analyzed by high-performance liquid chromatography(HPLC, Model: SCL-10AVP; Shimadzu, Kyoto, Japan) follow-ing Lu et al. (2004). Authentic squalene standard (Sigma-Aldrich) was used for identification and quantification. Withano-lides were also extracted according to Qin et al. (2010) and theresidue was dissolved in methanol and analyzed by HPLC follow-ing Singh et al. (2015). All standards of withanolides were fromNatural Remedies (Bangalore, India). The area of individualwithanolides was determined after normalizing with the peak areaof the internal standard, catharanthine (Sigma-Aldrich).

Sterol extraction and analysis

Total sterols were extracted and saponified according to Darnet& Rahier (2004). The extract was dried on Na2SO4 and evapo-rated to dryness and dissolved in 30 ll dichloromethane contain-ing 120 ng of estrone (Sigma-Aldrich). Sample (2 ll) wasinjected into a GC 7890B apparatus (Agilent Technologies,Wilmington, DE, USA), which consisted of a 7693 automatedsampler injection system, split/splitless injector, HP-5 fuse-silicacapillary coated with 5% phenylmethyl siloxane column(30 m9 0.320 mm, 0.25 lm film thickness) and a flame ioniza-tion detection (FID) controlled by the Agilent OPENLAB CDS(EZchrom edition) v.A.04.07 software. GC conditions were fol-lowed as described previously (Singh et al., 2015). Authenticcholesterol, campesterol, stigmasterol, b-sitosterol andcycloartenol were purchased from Sigma-Aldrich (Fig. S3). Forquantification, a standard curve for individual authentic standardwas generated and the relative response factor (RRF) for eachstandard was determined. Then the area of peaks correspondingto individual phytosterols was normalized with estrone. The RRFand concentration of estrone were used to determine the concen-tration of individual phytosterols using the respective normalizedpeak area. The concentrations of phytosterols were expressed aspercentage content relative to control.


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Chromatin immunoprecipitation assay

For chromatin immunoprecipitation (ChIP) assays, 10 lg ofp326::WsWRKY1-sGFP and p326::sGFP constructs were trans-fected into protoplasts prepared from W. somnifera leaves. Proto-plasts were harvested after 16 h and ChIP assays were conductedas described previously (Bowler et al., 2004; Saleh et al., 2008).Protoplasts were fixed with formaldehyde and chromatin was iso-lated and sheared into fragments ranging from 300 to 400 bp bysonication. Anti-GFP antibody (Roche) was used for immunopu-rification of WsWRKY1-GFP–DNA and GFP–DNA complexes.ChIP-qPCR was performed using immunoprecipitated DNAand input DNA with primers flanking the W-box binding site ofthe SQS and SQE promoters. Primers detecting enrichment ofboth SQS and SQE promoters lacking W-box were used as nega-tive controls. ChIP signals were compared between WsWRKY1-GFP–DNA and GFP–DNA, and the ChIP results are shown aspercentage of input DNA.

Bacterial growth curve, fungal infection and insect feedingassays

For bacterial growth curve assay, avirulent and virulent(DC3000) strains of Pseudomonas syringae were cultured in 5 mlof nutrient broth medium in a shaking incubator at 28°C.Overnight-grown bacterial samples were centrifuged and bacte-rial pellets were homogenized in 10 mM MgCl2 and cell densitywas calculated as 19 108 colony-forming units (CFU) ml�1atOD600 = 0.2 (avirulent) and 19 106 CFUml�1 atOD600 = 0.002 (virulent). Bacterial infiltration and cfu determi-nation, and fungal infection and insect feeding assays were per-formed according to Singh et al. (2015).

Statistical analysis

Average mean, SE and number of replicates used for each experi-ment were employed for statistical evaluation using theGraphPad QUICKCALC online software (http://www.graphpad.com/quickcalcs/ttest1.cfm). The statistical significance of differ-ences between control and treated samples were tested byunpaired Student’s t-test.

Results

Isolation and expression profiling ofW. somnifera WRKY1

In our previous study, nitrogen treatment enhanced the accumula-tion of phytosterols and withanolides, which were correlated withinduced expression of WsWRKY1 (expressed sequence tag (EST)accession number GR923578), indicating the possible role ofWsWRKY1 in the regulation of triterpenoids (Pal et al., 2017).Here, to further investigate the involvement of WsWRKY1 in theregulation of triterpenoid withanolide biosynthesis, its expressionwas evaluated in response to MeJA and salicin that are known toinduce withanolides accumulation in W. somnifera (Rana et al.,2014). It was found that WsWRKY1 transcripts were induced in

response to both MeJA and salicin during different periods oftreatment (Fig. 1a). Compared with the uninduced controls,MeJA treatment resulted in the gradual increase of WsWRKY1transcripts at 6 h and reached a maximum at 12 h (approximatelyfivefold): thereafter it decreased and remained constant (Fig. 1a).In contrast to this, under salicin treatment, WsWRKY1 exhibited agradual increase in its expression, peaking at 24 h (approximatelyfourfold) and was found slightly reduced at 48 h (Fig. 1a). Inaccordance with the induction of WsWRKY1 expression, there wasa correlation of withaferin A (major leaf withanolide) accumula-tion in response to both MeJA and salicin treatments (Fig. 1b).While MeJA treatment showed a gradual increase of withaferin Aaccumulation, reaching a maximum (2.8-fold) at 48 h, salicintreatment resulted in a gradual increase until 24 h (c. 4.8-fold) andreduced at 48 h (threefold). Between the two phytohormones,salicin treatment resulted in higher accumulation of withaferin Acompared with MeJA treatment (Fig. 1b). Further tissue-specificexpression analysis showed the highest expression of WsWRKY1 inleaf (c. 5.5-fold) followed by stem (approximately twofold) withlowest expression in root tissue (Fig. 1c).

Sequence analysis and subcellular localization

As the EST sequence of WsWRKY1 was lacking the 30 region,full-length gene sequence was amplified using a forward primer(50-ATGGAGAGAGTTAAAGGTTTGGAGAAAAAG-30) anda reverse oligo (dT)18 primer. Analysis of confirmed sequencerevealed that WsWRKY1 contained a 1023 bp ORF that encodesa protein of 341 amino acids (accession number MF092860)with a calculated molecular mass of 38.52 kDa. The WsWRKY1protein contained one putative WRKY domain (WRKYGQK)together with one unique zinc finger-like motif in the C-terminalregion (Fig. 2a), and belonged to the group III WRKY family ofplant TFs as per the classification described by Eulgem et al.(2000). Generally WRKYs of group III contain a zinc-fingerstructure with the form of C–X7–C–X23-H–X–C, butWsWRKY1 has a unique C-X7-C-X22-H-X-C zing-finger motif.In silico analysis of WsWRKY1 sequence using prediction pro-grams revealed that it has a nuclear localization signal(104KKRK107) (Fig. 2a), which is a general characteristic of TFs.To experimentally determine the subcellular localization, thecoding region of WsWRKY1 was fused to a GFP reporter gene,and the fusion construct was then transfected into C. roseus proto-plasts. Analysis of fluorescence obtained from protoplasts express-ing the WsWRKY1-GFP fusion protein clearly indicated anuclear localization, whereas the fluorescence from the GFP con-trol was distributed throughout the cell (Fig. 2b). Phylogeneticanalysis of WsWRKY1 with characterized WRKY proteinsinvolved in regulating plant specialized metabolism and stresstolerance showed its close similarity to group III WRKYsfrom A. annua (AaWRKY1), M. truncatula (MtWRKY109669),Oryza sativa (OsWRKY45 and OsWRKY89) and C. roseus(CrWRKY1) (Fig. 2c). Multiple sequence alignment of group IIIWRKYs revealed that WsWRKY1 shares highest sequence iden-tity with AaWRKY1 (42%) followed by MtWRKY109669(40%), OsWRKY, OsWRKY and CrWRKY1 (34%) (Fig. S4).



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http://www.graphpad.com/quickcalcs/ttest1.cfm

http://www.ncbi.nlm.nih.gov/nuccore/GR923578

http://www.ncbi.nlm.nih.gov/nuccore/MF092860

Transcriptional down-regulation ofWsWRKY1 by VIGSleads to stunted growth and reduced withanolides

In order to test the in planta role in regulation of withanolidebiosynthesis, WsWRKY1 was subjected to VIGS based on themethod used earlier in W. somnifera (Singh et al., 2015). Sur-prisingly, VIGS of WsWRKY1 caused stunted growth com-pared with empty vector (EV) control plants (Fig. 3a) and the

degree of WsWRKY1 silencing correlated with the stuntednessof the plant (Fig. 3a). Next, to determine the effect ofWsWRKY1 silencing on withanolides, leaves of similar devel-opmental stages exhibiting the typical viral infection pheno-type were collected for transcript and metabolite analyses.RT-qPCR analysis showed reduction of c. 75% of WsWRKY1expression in WsWRKY1-vigs leaves compared to the EV con-trols (Fig. 3b). Further, the effect of WsWRKY1 silencing onwithanolide accumulation was determined by subjecting themethanolic leaf extracts to HPLC, which revealed a drasticeffect on withanolide accumulation, with a significant reduc-tion in individual withanolides (Fig. 3c). Among the analyzedwithanolides, all except 12-deoxywithastromonolide and with-anolide A exhibited a significant decline in their accumulationin WsWRKY1-vigs leaves compared with EV controls(Fig. 3d). Withanoside IV and withanolide B exhibited thehighest decline (c. 88%) followed by withaferin A (76%), andwithanoside V (42%) (Fig. 3d).

Suppression ofWsWRKY1 by VIGS negatively affects sterolpathway genes resulting in reduced cycloartenol andphytosterols

Based on the observation that WsWRKY1 silencing negativelyaffected plant growth and withanolide accumulation (Fig. 3),we hypothesized that WsWRKY1 could be affecting thebiosynthesis of phytosterols, which are required for normalgrowth and development of plants (Bach, 1995; Schaller,2003) and as precursors for withanolide biosynthesis inW. somnifera (Lockley et al., 1976; Sangwan et al., 2008).This led us to look more closely at the expression of genesencoding enzymes involved in phytosterol biosynthesis. RT-qPCR analysis revealed that several genes of the upstreamMVA pathway and downstream sterol pathway were significantlydown-regulated, albeit at different levels in WsWRKY1-vigstissues compared to control (Fig. 4a). Among the analyzedMVA pathway genes, transcripts for 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR1and HMGR2) and HMG-CoA synthase (HMGS) displayed areduction of c. 75%, whereas transcripts for mevalonatekinase (MK) exhibited c. 38% reduction (Fig. 4a). Withrespect to genes encoding enzymes involved in the synthesisof triterpenoid pathway intermediates, farnesyl diphosphatesynthase (FPPS), SQS, and SQE displayed a reduction of c.58%, 65% and 46%, respectively (Fig. 4a). While the geneencoding CAS (an oxidosqualene cyclase involved in provid-ing flux towards sterol biosynthesis) was significantly down-regulated (c. 46%), genes for other oxidosqualene cyclaseslike b-amyrin synthase (BAS), lupeol synthase (LS) andlanosterol synthase (LAS) were not affected (Fig. 4a). Amongthe analyzed downstream sterol pathway genes, those encod-ing cycloeucalenol cycloisomerase (CEC1), D14-sterol reduc-tase (FK), C-7,8 sterol isomerase (HYD1), C-5 steroldesaturase (STE1), D24 sterol reductase (DWF1), sterol D7

reductase (DWF5), and C-22 sterol desaturase (CYP710A1)exhibited a reduction ranging from 30% to 67%, with

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Fig. 1 Effect of methyl jasmonate (MeJA) and salicin onWithania

somnifera WRKY1 expression and withaferin A accumulation. (a, b)Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)analysis ofWsWRKY1 expression (a) and high-performance liquidchromatography (HPLC) quantification of withaferin A (b) inW. somnifera

leaves treated with 500 lMMeJA and 1.5 mM salicin at different timeintervals. (c) Relative transcript abundances ofWsWRKY1 in differenttissues ofW. somnifera. Expression level is displayed as relative expressioncompared with untreated leaves. 18S rRNAwas used as an internalreference for normalization. The withaferin A level in the untreated controlwas set to 100% to determine its relative abundance in MeJA- and salicin-treated samples. The data shown are from three independent experiments.Student’s t-test: *, P < 0.05; **, P < 0.01. Error bars indicate mean� SE.





CYP710A1 and DWF5 showing the least and highest down-regulation. However, transcripts for sterol methyltransferase(SMT1) and obtusifoliol 14-demethylase (OBT) were not sig-nificantly affected in WsWRKY1-silenced samples (Fig. 4a).The most striking down-regulation of genes as a result of

WsWRKY1 silencing was found among MVA pathway genes.As there was down-regulation of several sterol pathway genesincluding SQS and CAS in WsWRKY1-vigs leaves, we deter-mined the effect of this down-regulation on cycloartenol andphytosterol accumulation. Quantification of results from GC

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Fig. 2 Sequence analysis and subcellular localization ofWithania somniferaWRKY1. (a) Amino acid sequence of WsWRKY1 showing the nuclearlocalization signal (in green), signature WRKY motif (in blue), and the characteristic zinc finger motif (underlined in red). (b) Subcellular localization ofWsWRKY1 in Catharanthus roseus protoplasts. Green fluorescent protein (GFP) fluorescence (green) and Chl autofluorescence (red) observed in afluorescence microscope are shown in upper and lower panels, respectively. Bars, 50 lm. (c) Phylogenetic relationship of WsWRKY1 with other plantWRKYs involved in the regulation of secondary metabolism and stress tolerance. The tree was constructed using MEGA v.6, and statistical reliability ofindividual nodes of the tree was assessed by bootstrap analyses with 1000 replicates. Names of sequences with blue and pink circles represent WRKYsinvolved in specialized metabolism and biotic/abiotic stress, respectively. WsWRKY1 characterized in this study is shown with a green circle. The WRKYproteins, their respective plant species, and GenBank accession numbers are as follows: AaWRKY1, Artemisia annua (FJ390842); AaGSW1 (WRKY),A. annua (KX465128); AtWRKY1, Arabidopsis thaliana (AF442389); AtWRKY12, A. thaliana (AF404857); AtWRKY17, A. thaliana (NP_565574.1);AtWRKY18, A. thaliana (NP_567882.1); AtWRKY23, A. thaliana (AY052647); AtWRKY33, A. thaliana (AK226301); AtWRKY40, A. thaliana(NP_178199.1); AtWRKY44, A. thaliana (NM_129282); AtWRKY48, A. thaliana (NP_191199.1); AtWRKY70, A. thaliana (NP_191199.1); CrWRKY1,C. roseus (HQ646368); CjWRKY1, Coptis japonica (AB267401); GaWRKY1, Gossypium arboreum (AY507929); GmWRKY, Glycine max (AJB84600.1);HbWRKY1, Hevea brasiliensis (JF742559); HbWRKY41, H. brasiliensis (GU372969); MdWRKY100577,Medicago truncatula (EU526033);MdWRKY100630,M. truncatula (EU526034); MdWRKY108715,M. truncatula (EU526035); MdWRKY109669,M. truncatula (EU526036); MdSTP,M. truncatula (HM622066); NbWRKY8, Nicotiana benthamiana (AB445392); OsWRKY13,Oryza sativa (EF143611); OsWRKY45,O. sativa (AK066255);OsWRKY53,O. sativa (AB190436); OsWRKY76,O. sativa (AK068337); OsWRKY89,O. sativa (AY781112); PqWRKY1, Panax quinquefolius(JF508376); PsWRKY1, Papaver somniferum (JQ775582); PcWRKY1, Petroselinum crispum (AAD55974.1); SlWRKY73, Solanum lycopersicum(NM_001247873); TcWRKY1, Taxus chinensis (JQ250831); TaWRKY, Triticum aestivum (APT42877); TcWRKY33, Theobroma cacao (EOY34631);VvWRKY2, Vitis vinifera (AY596466); WsWRKY1,W. somnifera (GR923578).



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NewPhytologist6

analysis revealed a significant reduction of all analyzedmetabolites (Fig. 4b) in WsWRKY1-silenced leaves, with stig-masterol exhibiting the highest reduction (42%), followed by

cycloartenol (44%), campesterol (39%), cholesterol (32%),and, with the lowest reduction, sitosterol (31%) (Fig. 4c).

Transient overexpression ofWsWRKY1 up-regulates phy-tosterol pathway genes resulting in elevated phytosterolsand withanolides

Silencing of WsWRKY1 resulted in negative regulation of sterolbiosynthetic genes, leading to reduced accumulation of phytos-terols and withanolides (Figs 3, 4). To investigate whetherWsWRKY1 overexpression positively regulates the expression oftriterpenoid biosynthetic genes leading to increased phytosteroland withanolide content, a transient overexpression assay was car-ried out in W. somnifera leaves by agroinfiltration. Analysis ofmRNA expression in W. somnifera leaves infiltrated withA. tumefaciens carrying the WsWRKY1 gene under the control ofthe CaMV 35S promoter showed increasedWsWRKY1 transcripts(c. 3.4-fold) 2 d postinfiltration (Fig. 5a). Subsequent analysis ofsterol biosynthetic genes showed up-regulation of all analyzedgenes, with HMGR2 and CYP710A1 exhibiting the highest (c.8.3-fold) and lowest (approximately twofold) values (Fig. 5a).The increased expression of sterol pathway genes resulted inenhanced accumulation of phytosterols (Fig. 5b). While campes-terol showed the highest increase (700%) followed by cycloartenol(400%), cholesterol (340%) and stigmasterol (260%), b-sitosterolexhibited the smallest increase (200%) (Fig. 5b). Further analysisof withanolide contents in WsWRKY1-overexpressing leavesshowed a significant increase in the accumulation of withaferin A(218%), withanolide A (207%), withanolide B (155%) and with-anoside V (140%) (Figs 5c, S5). However, there was a significantreduction (30%) in 12-deoxywithastramonolide.

WsWRKY1 binds to promoters of SQS and SQE

Silencing and overexpression of WsWRKY1 in W. somniferadown- and up-regulated the expression of SQS and SQE, respec-tively, suggesting the possible interaction of WsWRKY1 withpromoters of these genes. The promoters of W. somnifera SQSand SQE have recently been found to contain the W-box core ele-ment TGAC at �373 and �288 positions, respectively (Fig. S6)(Bhat et al., 2012; Razdan et al., 2013). To confirm that theWsWRKY1 physically interacts with SQS and SQE promoters, aChIP assay was carried out using W. somnifera protoplasts trans-formed with WsWRKY1–GFP and GFP constructs. ElutedChIPed DNA was analyzed by RT-qPCR using primers corre-sponding to SQS and SQE promoter regions. While there wasprofound enrichment in both SQS and SQE promoters withprimer pairs specific to W-box elements, there was no enrichmentwhen other distant sequences in the SQS and SQE promoterswere tested (Fig. 6a,b). This indicated the direct interaction ofWsWRKY1 with SQS and SQE promoters, which supported theregulation of corresponding genes in VIGS and overexpressionbackground. To further validate this, squalene was analyzed inleaves transiently overexpressing WsWRKY1. HPLC analysis andquantification showed a drastic increase of squalene (213%) com-pared with vector controls (Fig. 6c).

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Fig. 3 Tobacco rattle virus-mediated silencing ofWsWRKY1 and its effecton plant growth and withanolide content inWithania somnifera. (a)Phenotype ofW. somnifera plants exhibiting different degrees ofWsWRKY1 silencing. (b) Quantitative reverse transcription polymerasechain reaction (RT-qPCR) analysis ofWsWRKY1 expression; (c, d) high-performance liquid chromatography (HPLC) quantification of differentwithanolides in control andWsWRKY1-silenced leaves. Expression level ofWsWRKY1was normalized to 18S rRNA and set to 1 in an empty vector(EV) control to determine the relative reduction inWsWRKY1-silencedleaves. (c) Chromatograms showing relative withanolide content inWsWRKY1-silenced leaves compared with controls. Withanolides wereextracted from dried leaves and quantified by HPLC analysis. (d) Levels ofdifferent withanolides are represented relative to EV. The area of individualwithanolides was determined after normalizing with peak area of internalstandard (catharanthine). Wit-IV, withanoside IV; Wit-V, withanoside V;Wit(de)-A, withanolide A; Wit(de)-B, withanolide B; Wit(in)-A, withaferinA; 12d-wit, 12-deoxywithastramonolide. The results shown are from (a)three, (b) five and (d) 10 independent experiments. Student’s t-test: *,P < 0.05; ***, P < 0.001. Error bars indicate mean� SE.





WsWRKY1 silencing negatively affects the expression ofdefense genes, resulting in reduced tolerance to bioticstress

As WRKY proteins and phytosterols generally contribute to plantdefense, the effect of WsWRKY1 silencing on the expression ofdefense-related genes was determined by RT-qPCR. Among thegenes encoding pathogenesis-related (PR) proteins, bothSA-dependent PR1 (62%) and PR5 (74%), and jasmonic acid(JA)-dependent PR3 (58%) exhibited a drastic reduction in theirtranscript abundances in WsWRKY1-vigs samples compared withcontrols (Fig. 7a). It has been reported that expression of PR isdependent on nonexpressor of PR (NPR) (Fan & Dong, 2002),as well as brassinolide (Divi et al., 2010). Hence, we analyzed theeffect ofWsWRKY1 silencing on the expression of genes encodingNPR1 and BR6OX2 (which catalyzes the conversion of castas-terone to brassinolide) and observed a significant reduction ofNPR1 (44%) and BR6OX2 (67%) transcripts (Fig. 7a), indicativeof a link between the concentrations of endogenous sterols andbrassinolide and the level of NPR-dependent PR gene expression.The reduced gene expression of PR, NPR and BR6OX2 inWsWRKY1-silenced leaves was correlated with reduced toleranceto biotic stresses (Fig. 7). A bacterial growth assay using extractsisolated from P. syringae-infiltrated leaves showed thatWsWRKY1-silenced leaves supported a significantly higher

multiplication of both avirulent and virulent P. syringae strainsthan extracts from EV-infiltrated leaves (Fig. 7b). In fungal infec-tion assays with the necrotrophic pathogen Botrytis cinerea,WsWRKY1-vigs leaves developed severe disease symptoms andsustained more tissue damage than control leaves at 4 d postinoc-ulation (Fig. 7c). WsWRKY1-vigs leaves were chlorotic or macer-ated, whereas leaves from control plants remained green withnecrotic spots restricted to the initial culture droplet area, with-out much spreading of infection (Fig. 7c). WsWRKY1-vigs leavesexhibited reduced tolerance to B. cinerea infection, which was evi-dent by a significant increase (c. 2.71-fold) in the average diame-ter of lesions compared with that of EV leaves (Fig. 7d). Further,insect feeding assay using tobacco cutworms (Spodoptera litura)showed that the larvae preferred eating WsWRKY1-silenced leavesthan control leaves (Fig. 7e). Also, preference index (PI) determi-nation showed a higher preference of tobacco cutworm larvae forWsWRKY1-vigs leaves (PI = 1.58) compared with the EV control(PI = 1.0) (Fig. 7f).

Overexpression ofWsWRKY1 in tobacco positively regu-lates phytosterol biosynthesis and biotic stress

To check whether WsWRKY1 positively affects phytosterol anddefense response in a heterologous host system, transgenictobacco plants overexpressing WsWRKY1 (OE-WsWRKY1) were

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Fig. 4 Relative levels of mevalonate and sterol pathway gene expression, and phytosterols in control andWsWRKY1-silencedWithania somnifera leaves.(a) Expression levels of genes were normalized to 18S rRNA and are represented as expression relative to an empty vector (EV) control that was set to 1. (b)Sterol content changes inWsWRKY1-silenced leaves compared with EV control. Total phytosterols were extracted from dried leaves and quantified by GCanalysis. Total ion chromatograms (TIC) are shown. (c) Relative levels of phytosterols inWsWRKY1-silenced and control leaves. The peak area of individualphytosterols was normalized with the internal standard estrone, and the concentration of phytosterols in EV was set to 100 to determine the level inWsWRKY1-silenced leaves. The results shown are from three (a) and six (c) independent experiments. Student’s t-test: *, P < 0.05; **, P < 0.01. Error barsindicate mean� SE.



Research

NewPhytologist8

generated. The expression levels of three independent lines weredetermined by RT-qPCR. Plants transformed with the emptypBI121 vector served as controls. Transgenic OE-WsWRKY1lines showed expression ranging from 40- to 85-fold in analyzedlines (Fig. 8a). To investigate if the overexpression of WsWRKY1has any effect on the transcript abundance of sterol pathwaygenes, the mRNA expression of HMGR1, FPPS, SQS, SQE, CAS,OBT and SMT1 in OE-WsWRKY1 lines was analyzed. Resultsindicated that all selected pathway genes were up-regulated, albeitat different levels, in all OE-WsWRKY1 lines as compared withthe pBI121 control (Fig. 8b). The up-regulation of sterol path-way genes in OE-WsWRKY1 could have a positive regulatoryeffect on phytosterol accumulation similar to the results obtainedin W. somnifera (Fig. 5a,b). Hence, phytosterols were analyzedusing leaves of OE-WsWRKY1 plants and control tobacco plants,which showed a significant increase in sterol intermediatessqualene and cycloartenol (Fig. 8c,d). With respect to phytos-terols, except for b-sitosterol, which showed a significant reduc-tion in all OE-WsWRKY1 lines, all other phytosterols exhibited asignificantly increased accumulation (Fig. 8d). To determine theeffect of WsWRKY1 overexpression on biotic stress, a bacterialgrowth assay was performed, which revealed a significant toler-ance of OE-WsWRKY1 samples to P. syringae (strain DC3000)growth compared with controls (Fig. 8e).

Discussion

In this study, we set out to investigate the transcriptional regula-tion of triterpenoid withanolide biosynthesis in W. somnifera.Our previous study found that a particular TF belonging to theWRKY class (WsWRKY1) was highly induced in response tonitrogen treatment, with a corresponding increase in withano-lides (withaferin A) as well as phytosterols (Pal et al., 2017).Here, a series of investigations was carried out to further deter-mine the functional role of WsWRKY1. Phytohormones havebeen shown to up-regulate the expression of pathway genes andWRKY TFs involved in secondary metabolism with a corre-sponding increase in target secondary metabolites (Ma et al.,2009; Suttipanta et al., 2011; Chen et al., 2016). InW. somnifera,the expression of SQS, SQE and CAS, involved in phytosterolbiosynthesis, was highly induced by MeJA and SA, with a corre-sponding increase in withanolides (Bhat et al., 2012; Dhar et al.,2014). Moreover, RNAi and homologous overexpression of CASresulted in a respective reduction and enhancement of withano-lide accumulation in W. somnifera plants (Mishra et al., 2016). Inthis study, WsWRKY1 exhibited significant up-regulation inresponse to MeJA and salicin (Fig. 1a), and its expression wascorrelated with accumulation of withaferin A, the major withano-lide in leaves (Fig. 1b). WsWRKY1 up-regulation in response to

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Fig. 5 Effect of transient overexpression ofWsWRKY1 on mevalonate and phytosterol pathway genes, phytosterols, and withanolides inWithania

somnifera. (a) Relative levels ofWsWRKY1, mevalonate and sterol pathway genes in leaves infiltrated with Agrobacterium harboring pBI121 control andpBI121::WsWRKY1 overexpression constructs. Expression levels of genes were normalized to 18S rRNA and are represented as expression relative to apBI121 control that was set to 1. (b) Phytosterols were extracted from dried leaves and quantified by GC analysis. The peak area of individual phytosterolswas normalized with the internal standard estrone and expressed as relative percentage to the pBI121 control. (c) Levels of individual withanolides arerepresented relative to the pBI121 control. The area of individual withanolides was determined after normalizing with the peak area of the internalstandard. Wit-V, withanoside V; Wit(de)-A, withanolide A; Wit(de)-B, withanolide B; Wit(in)-A, withaferin A; 12d-wit, 12-deoxywithastramonolide. Theresults shown are from three to four (a), four (b) and six (c) biological replicates. Student’s t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error barsindicate mean� SE.





phytohormones and its high abundance in leaves indicated itspossible role in regulation of withanolide biosynthesis inW. somnifera (Fig. 1c).

To determine the involvement of WsWRKY1 in withanolidebiosynthesis, a reverse-genetics VIGS approach was used todown-regulate WsWRKY1 in W. somnifera leaves. Silencing ofWsWRKY1 resulted in stunted growth of the plant with a signifi-cant decrease in withanolide content (Fig. 3). The morphogeneticinhibition and a reduced withanolide content caused byWsWRKY1 silencing in W. somnifera were similar to previousreports in which down-regulation/knockout of sterol pathway

genes such as HMGR (Suzuki et al., 2004), CAS1 (Gas-Pascualet al., 2014), SMT (Choe et al., 1999), FK (Schrick et al., 2000)and OBT (Burger et al., 2003) led to reduced phytosterols andseverely stunted growth in different plants. Hence, we hypothe-sized that the effect of WsWRKY1 silencing on both withanolidesand plant growth in W. somnifera could be a result of the deple-tion of sterol precursors, which are required for withanolide for-mation (Singh et al., 2014) as well as for normal growth anddevelopment of the plant (Bach, 1995; Schaller, 2003). Indeed,WsWRKY1-silenced leaves exhibited significant down-regulationof several genes of the phytosterol pathway, including genes ofthe upstream MVA pathway, and the first committed step oftriterpene formation (SQS) (Wu et al., 2012) (Fig. 4a). Interest-ingly, among the genes for branch point oxidosqualene cyclases,only CAS exhibited significant down-regulation similar to down-stream sterol pathway genes, whereas BAS, LAS and LS wereunaffected by WsWRKY1 silencing (Fig. 4a). This indicated thespecific regulation of sterol branch of the triterpenoid pathway byWsWRKY1. Such differential regulation between distinct parts ofthe triterpenoid pathway has recently been reported inM. truncatula by bHLH family TFs (TRITERPENE SAPONINBIOSYNTHESIS ACTIVATING REGULATOR, TSAR1 andTSAR2) (Mertens et al., 2016), and in tomato by theAPETALA2/ERF GLYCOALKALOID METABOLISM 9(GAME9) (C�ardenas et al., 2016) and a jasmonate-responsiveERF (JRE) (Thagun et al., 2016) (Fig. S7). Transcript reductionof sterol pathway genes byWsWRKY1 silencing was accompaniedby a significant reduction in the concentrations of cycloartenoland phytosterols (Fig. 4c). This suggested that WsWRKY1 con-trols the flux towards sterol and withanolide biosynthesis throughthe regulation of several pathway steps. Recently, determinationof the relative importance of transcriptional control at each indi-vidual step of isoprenoid precursor biosynthesis showed that the‘flux control’ in the sterol pathway is shared by several enzymes(Lange et al., 2015). Specialized metabolites are generally formedfrom primary metabolites (Laursen et al., 2015); hence reducedwithanolide (specialized metabolite) accumulation inWsWRKY1-silenced plants could be a result of reduced phytosterols (primarymetabolites). Alternatively, reduced withanolides might be aresult of down-regulation of yet to be identified genes of thewithanolide pathway by WsWRKY1 silencing, which is corrobo-rated by the more drastic reduction of withanolides than of phy-tosterols (Figs 3, 4). This was supported by the fact that theexpression of genes encoding CYP450 enzymes (WsCYP93Id,WsCYP76A and WsCYP98A) and GTs (WsSGT1, WsSGT2and WsSGT4), which are indicated to be involved in withanolideformation (Rana et al., 2014; Srivastava et al., 2015; Singh et al.,2016), was drastically reduced in WsWRKY1-silencedW. somnifera leaves (Fig. S8).

In contrast to WsWRKY1-vigs, transient overexpression ofWsWRKY1 in W. somnifera leaves up-regulated phytosterol path-way genes such as HMGS, HMGR, SQS, SQE, FK, DWF1 andDWF5 (Fig. 5), and withanolide pathway genes such asWsCYP93Id, WsCYP76A, WsCYP98A, WsSGT1, WsSGT2 andWsSGT4 (Fig. S8), thus leading to enhanced phytosterol andwithanolide content (Fig. 5). This supported the observation

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Fig. 6 Binding of WsWRKY1 toWsSQS andWsSQE promoters, and effectofWsWRKY1 overexpression on squalene accumulation. (a) Schematicdiagram showing the location of W-box core elements (indicated asvertical lines) in promoter sequences ofWsSQS andWsSQE. A and Bindicate the location of primers corresponding to promoter regions ofWsSQS andWsSQE used in chromatin immunoprecipitation (ChIP)coupled with quantitative PCR (ChIP-qPCR) analysis. (b) ChIP assay ofWsWRKY1.Withania somnifera leaf protoplasts were transfected withWsWRKY1-GFP and GFP constructs. WsWRKY1-GFP and GFP chromatincomplexes were immunoprecipitated using anti-GFP antibody. ChIP andinput DNA samples were quantified by qPCR using specific primers A andB. The ChIP results are shown as a percentage of input DNA. Datarepresent three independent experiments. Student’s t-test: **, P < 0.01. (c)Withania somnifera leaves were infiltrated with Agrobacterium harboringpBI121 control and pBI121::WsWRKY1 overexpression constructs, andtissue was collected after 48 h. Squalene was extracted from dried leavesand analyzed by high-performance liquid chromatography andrepresented as relative levels. The data shown are from three independentexperiments. Student’s t-test: **, P < 0.01. Error bars indicate mean� SE.



Research

NewPhytologist10

made in VIGS studies that WsWRKY1 regulates phytosterol andwithanolide biosynthesis, suggesting the possible in planta inter-action of WsWRKY1with promoters of genes involved in phytos-terol and withanolide biosynthesis. As WRKY proteins bind toW-box elements (Eulgem et al., 2000) to regulate the geneexpression, it is possible that W-boxes (TTGACC/T) could bepresent in promoters of MVA and sterol pathway genes inW. somnifera. Indeed, promoter regions of SQS and SQE containW-box in W. somnifera (Bhat et al., 2012; Razdan et al., 2013).The ChIP-qPCR assay showed that WsWRKY1 binds to pro-moters of SQS and SQE (Fig. 6b), suggesting their direct regula-tion by the WsWRKY1. This was further supported by increasedaccumulation of squalene when WsWRKY1 was transiently over-expressed in W. somnifera leaves (Fig. 6c). Interestingly, althoughthe promoter of CAS lacks a W-box (Dhar et al., 2014), itsexpression was down- and up-regulated in WsWRKY1 silencingand overexpression background, respectively, which may be a

result of the indirect effect of reduced and increased transcripts ofseveral genes upstream of CAS (Figs 4a, 5a). It was reported ear-lier that overexpression of HMGS (Wang et al., 2012) and silenc-ing of SQS (Singh et al., 2015) result in corresponding up- anddown-regulation of other sterol pathway genes, indicating a tightregulation of sterol pathway genes. However, at this point, thepossibility of direct binding of WsWRKY1 to promoters of phy-tosterol pathway genes other than SQS and SQE cannot be ruledout in W. somnifera. Moreover, ectopic expression of WsWRKY1in tobacco not only up-regulated sterol pathway genes but alsoincreased the phytosterol content (Fig. 8), demonstrating thatWsWRKY1 is also capable of regulating sterol pathway in otherplant species. Also, WsWRKY1 overexpression in tobacco led tofaster growth and improved plant phenotype in terms of plantheight (Fig. S9), which was in contrast to the negative effect ofWsWRKY1-vigs on W. somnifera growth. Further, a transientoverexpression of WsWRKY1 in the crop plant, tomato, resulted

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Fig. 7 Effect ofWsWRKY1 silencing on defense-related genes and biotic stress inWithania somnifera. (a) Expression levels of genes were normalized to18S rRNA and are represented as expression relative to the empty vector (EV) control, which was set to 1. (b) Bacterial growth assay with Pseudomonassyringae. The first pair of EV andWsWRKY1-vigs leaves at 30 d postinfiltration (dpi) displaying a viral infection phenotype was infiltrated with avirulentand virulent (DC3000) strains of P. syringae. Bacterial growth at 4 dpi was obtained by plating serial dilutions of leaf extracts from EV andWsWRKY1-vigs.(c) Phenotype of Botrytis cinerea infection 4 d postinoculation on detached leaves of EV control andWsWRKY1-vigs leaves. The first pair of EV andWsWRKY1-vigs leaves at 30 dpi having viral infection phenotype was inoculated with a 20 ll drop of Botrytis cinerea spore suspension (c. 105 spores ml�1)at the center of the abaxial side and kept at 23°C with high humidity. (d) Statistical data of lesion size at 4 dpi. (e) Insect feeding assay of EV andWsWRKY1-vigs leaves using tobacco cutworm (Spodoptera litura). Equal numbers of second-instar cutworms (six each) were placed on EV andWsWRKY1-vigs leaves, and the area fed was recorded after 12 h. (f) Feeding preference index (PI) of tobacco cutworms. The eaten area of leaf wasmeasured 12 h after the start of the experiment to calculate the PI. The presented data are from three independent experiments. Student’s t-test:*, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars indicate mean� SE.





in improved formation of squalene and phytosterols (Fig. S10),indicating that WsWRKY1 can be a promising candidate forenhancing nutraceutically important phytosterols in tomatofruits. These results comprehensively prove that WsWRKY1 isinvolved in regulation of phytosterol biosynthesis, which in turn

controls withanolide accumulation in W. somnifera. Given thatTFs are often capable of coordinating the transcription of multi-ple biosynthetic pathway genes (Grotewold, 2008), it is not sur-prising that WsWRKY1 affects several genes of the MVA andphytosterol pathways. Similar coordinated up-regulation of

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**

**

**

*****

********

*****

OE-WsWRKY1-1OE-WsWRKY1_2OE-WsWRKY1_3

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8

8.1

8.2

8.3

*

**

**

OE-

WsWRKY1_1

OE-

WsWRKY1_2OE-

WsWRKY1_3

pBI121

WsWRKY1

17 18 19 20 21 22 23 24 25 26

Rela

�ve

abun

danc

e (T

IC)

OE- WsWRKY1_2

pBI121

Cholesterol

Campesterol

S�gmasterol

β-sitosterol

Cycloartenol

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

HMGR1 FPPS SQS SQE CAS SMT OBT

pBI121

OE-WsWRKY1

OE-WsWRKY2

OE-WsWRKY3

pBI121

**

****

OE-WsWRKY1_1OE-WsWRKY1_2OE-WsWRKY1_3

**

**

*

**

***

***

**

**

**

******

*

***

***

*

*

log

CFU

cm

–2

0

(a)

(c)

(d) (e)

(b)

Fig. 8 Analysis of phytosterol pathway gene expression, triterpenoid content, and bacterial growth in transgenic tobacco overexpressingWsWRKY1.Expression ofWsWRKY1 (a) and sterol (b) pathway genes in transgenic tobacco (Nicotiana tabacum) lines. Expression levels of genes were normalized toEF-1a and are represented as expression relative to the pBI121 control, which was set to 1. (c) Sterol content changes in OE-WsWRKY1 leaves comparedwith control. Phytosterols were extracted from dried leaves and quantified by GC analysis. Total ion chromatograms are shown. (d) Relative concentrationsof phytosterols in OE-WsWRKY1 and control leaves. The peak area of individual phytosterols was normalized with the internal standard estrone andexpressed as relative percentage to the pBI121 control. (e) Bacterial growth assay was performed by infiltrating leaves of pBI121 and OE-WsWRKY1withthe virulent P. syringae (DC3000) strain. Bacterial growth at 3 d post infiltration (dpi) was obtained by plating serial dilutions. The data presented here arefrom three (a, b), four (d), and three (e) independent experiments. Student’s t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars indicate mean� SE.



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NewPhytologist12

biosynthetic genes of secondary metabolism by WRKYs has beenobserved in other plant species. For example, overexpression of atrichome-specific WRKY in A. annua (AaGSW1) up-regulatedthe genes of the artemisinin pathway, resulting in improved accu-mulation of artemisinin (Chen et al., 2016). Similarly,CrWRKY1 in C. roseus positively regulated the expression of

several terpene indole alkaloid (TIA) pathway genes, therebyimproving the biosynthesis of TIAs (Suttipanta et al., 2011).Moreover, analysis of recently released genome sequence ofSolanaceae plants such as tomato and potato revealed the pres-ence of W-box sequences in MVA and sterol pathway genes(Table S2), suggesting a similar scenario inW. somnifera.

Ac-CoA + AcAc-CoA

HMG-CoA

HMGS

MVA

HMGR

MVA-5-P

MK

IPP

FPP

Squalene

SQS

DMAPP

FPPS

2,3-oxido squalene Cycloartenol

CAS SQE

Lanosterol

LAS

Cholesterol

SMT1

Cycloeucalenol

SMO1

Obtusifoliol

CECI

OBT FK

4α-methyl fecosterol

HYDI 24-methylene lophenol

SMO2 Episterol

β-amyrin

∆8,14 sterol

24-methylene cholesterol

STE1

Isofucosterol Sitosterol

DWF1

Stigmasterol

CYP710A1

Campesterol

DWF1

Castasterone BR6OX2

Brassinolide

24-methylene cycloartenol

5-dehydro episterol

DWF5

BAS

Withanolides

MVA

pat

hway

IPPI

Cytosol

Sesqui- terpenes

Lupeol

MEP pathway

DMAPP IPP IPPI

Plastid

Fig. 9 Simplified view of isoprenoid biosynthesis in plants. Solid black arrows indicate single-step reactions, dashed arrows denote several steps, dottedarrows represent unidentified steps and the double arrow between cytosolic and plastid compartments indicates metabolic crosstalk between them. Down-regulation of genes as a result ofWsWRKY1 silencing inWithania somnifera is indicated by downward red arrows, and up-regulation of genes inW. somnifera and tobacco overexpressingWsWRKY1 is shown as upward blue and purple arrows, respectively. Red hyphens indicate no change inexpression level as a result ofWsWRKY1 silencing inW. somnifera.Metabolites that are quantified in this study are shown in yellow. Ac-CoA, acetyl CoA;AcAc-CoA, acetoacetyl CoA; BAS, b-amyrin synthase; BR6OX2, brassinosteroid-6-oxidase 2; CAS, cycloartenol synthase; CECI, cycloeucalenolcycloisomerase; CYP710A1, C-22 sterol desaturase; DMAPP, dimethylallyl diphosphate; DWF1, D24 sterol reductase; DWF5, sterol D7 reductase; FK, D14-sterol reductase; FPP, farnesyl diphosphate; FPPS, FPP synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, HMG-CoA reductase; HMGS,HMG-CoA synthase; HYDI, C-7,8 sterol isomerase; IPP, isopentenyl diphosphate; IPPI, isopentenyl diphosphate isomerase; LAS, lanosterol synthase; LS,lupeol synthase; MEP, 2-C-methyl-D-erythritol 4-phospahte; MVA, mevalonate; MVA-5-P, 5-phosphomevalonate;MK, mevalonate kinase;OBT,obtusifoliol 14-demethylase; SMO1, sterol-4a-methyl oxidase 1; SMO2, sterol-4a-methyl oxidase 2; SMT1, sterol methyltransferase 1; SQE, squalenemonooxygenase/epoxidase; SQS, squalene synthase; STE1, C-5 sterol desaturase.





WRKY TFs are generally known to be responsive to pathogeninfection and phytohormone treatment, and to act as positive ornegative regulators of plant defense responses (Schluttenhofer &Yuan, 2015). Suppression of WsWRKY1 in W. somnifera nega-tively affected defense responsive genes (Fig. 7a), which was simi-lar to the effect of WsSQS silencing (Singh et al., 2015).Consistent with the MeJA- and SA-induced expression ofWsWRKY1 in W. somnifera (Fig. 1a), both SA-dependent PR1and PR5 (thaumatin) and JA-dependent PR3 (chitinase) expres-sion were down-regulated in WsWRKY1-silenced plants (Fig. 7a).WsWRKY1-vigs plants also showed drastic reductions of BR6OX2mRNA levels (Fig. 7a), suggesting possible reduction in endoge-nous brassinolide concentrations. Moreover, NPR1, a master reg-ulator of PR genes (Fan & Dong, 2002), was also significantlyreduced in WsWRKY1-vigs plants (Fig. 7a). Reduction ofdefense-related genes (PR) and their regulators (NPR1, BR60X2)could be a result of low transcript abundance of WsWRKY1,thereby resulting in reduced expression of defense-related genesand their regulators, which are known to be a direct target ofWRKY TFs, owing to the presence of WRKY binding site(s) (W-box) in their promoters (Asai et al., 2000; Yu et al., 2001). Theseobservations were further supported by reduced tolerance ofWsWRKY1-vigs plants to bacteria P. syringae and fungusB. cinerea infection, and tobacco cutworm feeding (Fig. 7). Spe-cialized metabolites regulated by WRKY TFs contribute to over-all fitness of the plant by enhancing tolerance to various stresses(Schluttenhofer & Yuan, 2015). Withanolides have been shownto possess antibacterial and antifungal properties (Choudharyet al., 1995), and antifeedant properties against herbivorousinsects (Waiss et al., 1993). Hence, it seems likely that reducedconcentrations of withanolides could have compromised the tol-erance of WsWRKY1-vigs leaves to bacterial and fungal infection,and to insect feeding. Furthermore, reduced NPR1 expressionmay also have contributed to the reduced tolerance ofWsWRKY1-vigs leaves to cutworm feeding. It was shown thatNPR1-silenced Nicotiana attenuata plants were more susceptibleto Spodoptera exigua larvae, suggesting NPR1’s role in mediatingherbivore resistance in plants (Rayapuram & Baldwin, 2007). Incontrast to the negative effect of WsWRKY1 silencing on bacterialstress in W. somnifera (Fig. 7b), transgenic tobacco overexpressingWsWRKY1 displayed an enhanced tolerance to P. syringae(Fig. 8e), confirming the involvement of WsWRKY1 in bioticstress tolerance.

Overall, WsWRKY1 regulated the expression of MVApathway genes and downstream sterol pathway genes, result-ing in corresponding regulation of phytosterols and withano-lides (Fig. 9). Studies on TFs such as M. truncatula TSARs(Mertens et al., 2016), tomato GAME9 and JRE (Thagunet al., 2016; C�ardenas et al., 2016) along with studies on theinvolvement of W. somnifera WRKY1 (this work) intriterpenoid pathway regulation, indicate that variousbranches of the pathway are differentially regulated by TFsbelonging to divergent classes, further highlighting the com-plex regulation of terpenoid biosynthesis in plants. It is pos-sible that these TFs interact in combination to bring aboutthe required accumulation of target metabolites, as has been

reported for terpene indole alkaloid biosynthesis in C. roseus(Paul et al., 2016). The presence of WsWRKY1 homologs inother solanaceae plants (such as tomato, potato and cap-sicum; Fig. S11) further suggests that these homologs mightbe functioning in a similar fashion to regulate the phytos-terol branch of the triterpenoid pathway in these species aswell. Overall, these findings demonstrate the importance ofWsWRKY1 in the regulation of W. somnifera phytosterol andwithanolide biosynthesis in particular, and plant phytosterolbiosynthesis and defense in general. Hence, WsWRKY1 couldbe useful for engineering enhanced withanolide biosynthesisin W. somnifera and improved phytosterol and biotic stresstolerance in solanaceous plants.

Acknowledgements

This work was supported by the BSC-0203 project of 12th FYPto CSIR-CIMAP. A.K. Singh, V.D. and A.R. are the recipientsof research fellowships from the Council of Scientific and Indus-trial Research (CSIR) and the University Grants Commission(UGC), respectively. S.R.K. is the recipient of CSIR seniorresearch associateship. The authors express their sincere gratitudeto Prof. Anil Kumar Tripathi, Director, CSIR-CIMAP, for hissupport throughout the study. We are also thankful to DrsInhwan Hwang, Boris Vinatzer, Ravi Manjithaya, and S.G.Eswara Reddy, for sharing p326-sGFP, P. syringae DC3000,anti-GFP antibodies, and tobacco cutworm eggs, respectively.We also thank Krishna Kumar and Seema Meena for their helpin VIGS and transgenic plant analysis. Special thanks go to Prof.Thomas J. Bach and Dr Michael Long for their critical readingand suggestions to improve the manuscript. No conflict of inter-est is declared. The institutional communication number for thisarticle is CIMAP/PUB/2017/3.

Author contributions

A. K. Singh., S.R.K., V.D., A.R. and S.P. performed the experi-ments. A. K. Singh, S.R.K., V.D., A.R., S.P., A. K. Shasany andD.A.N. analyzed the data. D.A.N. conceived and coordinatedthe research, and wrote the manuscript.

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Supporting Information

Additional Supporting Information may be found online in theSupporting Information tab for this article:

Fig. S1 p326-sGFP, pTRV2 and pBI121 derived vector mapsused for subcellular localization, VIGS and overexpression stud-ies, respectively.

Fig. S2 Expression analysis of WsWRKY1 and two other similarWRKY genes inWsWRKY1-vigs leaves.

Fig. S3 GC chromatogram showing authentic sterol standards.

Fig. S4 Multiple sequence alignment of WsWRKY1 with othergroup III WRKYs involved in secondary metabolism.

Fig. S5 HPLC chromatograms showing relative abundance ofwithanolides in W. somnifera infiltrated with pBI121 andpBI121::WsWRKY1 constructs.

Fig. S6 Promoter sequences of genes encoding SQS, SQE andCAS in W. somnifera, indicating the presence or absence of con-served W-box elements.



Research

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Fig. S7 Differential regulation of distinct parts of the triterpenoidpathway in M. truncatula, tomato, W. somnifera and tobacco bydifferent transcription factors.

Fig. S8 Effect of silencing and transient overexpression ofWsWRKY1 on genes encoding CYP450s and GTs indicated to beinvolved in withanolide biosynthesis inW. somnifera.

Fig. S9 Representative images showing the effect of WsWRKY1overexpression in tobacco seedlings, young plants and matureplants.

Fig. S10 Effect of transient overexpression of WsWRKY1 onsqualene and phytosterols in tomato leaves.

Fig. S11 Phylogenetic tree showing the relationship betweenWsWRKY1 and putative WRKY sequences from other solana-ceous plants.

Table S1 List of oligonucleotide primers used in this study

Table S2 Position of W-box in promoters of selected MVA andsterol pathway genes of Nicotiana tabacum and tomato

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A WRKY transcription factor from Withania somnifera...

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